Abstract Miscarriage causes termination of 15-20% of implantation-confirmed pregnancies. Majority of miscarriages occur during the first trimester of pregnancy and defective development of trophoblast cell lineage, which assures embryo implantation and placentation, is one of the leading causes for early pregnancy loss. However, we have a poor understanding of molecular mechanisms that regulate trophoblast progenitor differentiation and function in early post-implantation embryos. Our published and preliminary studies establish that GATA family transcription factors, GATA2 and GATA3, are conserved in trophoblast progenitors across mammalian species and are critical regulators to specify and maintain trophoblast lineage during early mammalian development. The overarching goal of this proposal is to further define GATA factor- mediated transcriptional mechanisms that regulate development and proper function of specialized trophoblast cell types at the maternal-fetal interface. Three specific aims are proposed. Aim 1 will study a Gata2/Gata3 double knockout (Gata2-/-/Gata3-/-) mouse model and GATA-mutated rat models to test the hypothesis that cell-autonomous functions of GATA factors are essential to establish differentiated cell-fate in the trophoblast progenitors of a developing placenta. Aim 2 will perform global gene expression analyses in primary trophoblast cells of developing placenta with or without GATA mutations. The goal is to test the hypothesis that GATA factors establish differential transcriptional circuitry in trophoblast stem/progenitor cells vs. differentiated trophoblast cells, thereby specifying trophoblast subtypes. Aim 3 will study GATA-mediated regulation of gene expression in human trophoblast cells. The objective is to test the hypothesis that GATA2 and GATA3 are essential to maintain a core TSC-specific transcriptional program in trophoblast progenitors across mammalian species, including human villous cytotrophoblast (vCTB) progenitors of a human placenta. We will also test whether pregnancy associated disorders involve altered GATA factor-mediated gene regulation in vCTBs.